Viral gene transfer vectors are widely used throughout molecular biology, especially in gene therapy. A major problem posed by the use of viral gene transfer vectors for gene therapy is the lack of consistent and effective methods to assess the purity of a stock of viral gene transfer vector. The lack of efficient means for determining purity of viral gene transfer vectors has resulted in the inability to measure the effectiveness or consistency of viral gene transfer vector production techniques. These problems are magnified when considering the safety concerns surrounding use of viral gene transfer vectors in human gene therapy.
Traditionally, attempts to measure the purity of an unmodified virus particle in a medium have relied on electrophoresis techniques, such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or ultraviolet (UV) spectroscopy measurements. Capillary gel electrophoresis also has been used to analyze viral components in comparison to known standards, as illustrated in U.S. Pat. No. 5,723,031 (Durr et al.) In addition, ultraviolet spectroscopy techniques and ultraviolet resonance spectra have been used to provide structural analyses of several viruses.
Other techniques for assessing viral purity have centered on the use of chromatography techniques such as ion exchange chromatography, gel-filtration chromatography, and reverse phase high performance liquid chromatography (HPLC). For example, researchers have utilized HPLC to purify structural proteins from Herpes Simplex Virus type-1. Similar techniques for purifying viral components have been developed using cation exchange chromatography as described in U.S. Pat. No. 5,486,470 (Darke et al.), ion exchange chromatography as described in U.S. Pat. No. 5,696,238 (Haigwood and Scandella), and electrical chromatography as described in U.S. Pat. No. 5,707,850 (Cole and Gaithersburg).
Mass spectrometry, with the advantage of fast, accurate and detailed analysis as compared to several of the aforementioned techniques, has been used to study individual organic compounds. For example, Vastola et al., Organic Mass Spectrometry, 3, 101-104 (1969), disclosed the use of laser pyrolysis mass spectrometry to study the spectra generated by organic salts. Since that time, mass spectrometry has been widely used to evaluate features of larger organic molecules such as proteins. Recently these techniques have been used to study or validate the purity of individual viral proteins or groups of viral proteins such as the components and modifications of viral capsid proteins. For example, Fetzer et al., Protein Expression and Purification, 5, 432-441 (1994), teach the use of mass spectrometry in combination with SDS-PAGE to validate the purity of a herpes simplex virus thymidine kinase protein that was subjected to proteolytic cleavage and chromatography purification.
Other researchers have studied groups of viral proteins, for example, Davidson and Davidson, Virology, 206, 1035-1043 (1995), disclosed the identification of proteins of channel catfish virus by use of fractionation techniques in combination with laser desorption ionization mass spectrometry. Similarly, Pepinksy et al., J. Virology, 70, 3313-18 (1996), used electrospray mass spectrometry to obtain molecular masses for Gag proteins in the Rous sarcoma virus, to study protein intermediates, and to determine that exopeptidase trimming occurred in some of the Gag proteins.
More recently, Bothner et al., J. Biol. Chem., 273, 673-76 (1997), have disclosed the use of matrix assisted laser desorption/ionization mass spectrometry (MALDI or MALDI-MS), in combination with limited proteolysis, upon flock house virus particles to study viral capsid dynamics in that species. Also recently, mass spectrometry has been used as a tool for diagnosing specific DNA molecules in viruses, as described in U.S. Pat. No. 5,605,798 (Koster).
Not until very recently has mass spectrometry been applied to entire virus particles. Tas et al., Biomedical and Environmental Mass Spectrometry, 18, 757-760 (1989), met with mixed results in attempting to characterize Vero cell cultures infected with herpes simplex virus and poliomyelitis virus by studying low-weight molecules (i.e., salt molecules, methyl groups, and sugars) that were detected by use of pyrolysis/direct chemical ionization (Py/DCI) mass spectrometry. While Tas et al. suggested that they could possibly identify cell lines infected with a particular virus, they failed to teach a method of obtaining relatively pure viral stocks, especially of a viral gene transfer vector capable of use in molecular biological applications, particularly gene therapy.
Despeyroux et al., Rapid Communications in Mass Spectrom., 10, 937-41 (1996), were able to generate a spectrum for purified cricket paralysis virus (CrPV) by using electrospray ionization mass spectrometry and suggested that comparisons could be made to such spectra. However, Despeyroux et al.'s results are limited because success of their technique was attributed to the small size and simplicity of structural polypeptides of the cricket paralysis virus. Furthermore, similar to Tas et al., Despeyroux et al. failed to teach the combination of such techniques with a useful viral gene transfer vector.
Siuzdak et al., Chemistry & Biology, 3, 45-48 (1996), similarly used electrospray ionization (ESI) mass spectrometry, under special settings, to detect viruses. Using standard ESI techniques, Siuzdak et al. were not able to derive signals for large virus particles. Siuzdak et al. were capable of generating signals and performing mass selection on the charged particles from tobacco mosaic virus (TMV) and rice yellow motile virus (RYMV), by using the radio frequency (RF) mode of an ESI mass spectrometer in a manner that retained the ultrastructure of the virus particles. Thus, Siuzdak et al. taught that non-destructive ESI could actually purify virus particles. However, Siuzdak et al. only reported on small viruses (i.e., molecular weights of less than 50,000,000 Daltons), and did not assess the relative purity of a virus that had not been modified by subjection to mass spectrometry.
Siuzdak, J. Mass Spectrom., 33, 203-211 (1998), also disclosed that TOF mass spectrometry might be used to measure large ions such as a whole Tobacco Mosaic Virus. However, such work, if carried out, was not published, and nevertheless would not have provided a method of easily and efficiently assessing relative viral purity, especially in regard to large virus particles, such as with a stock of a useful viral gene transfer vector.
Moreover, many specific types of viral gene transfer vectors that have proven to be powerful tools in molecular biology often have molecular weights well in excess of those of TMV and RYMV. For example, adenoviral vectors, which are useful delivery vehicles, such as in gene therapy, have molecular weights often exceeding 150,000,000 Daltons, three times the weight of the viruses studied by Siuzdak et al.
Furthermore, the techniques taught by Tas et al., Despeyroux et al., and Siuzdak (i.e., ESI and Py/DCI mass spectrometry) are difficult to perform, and require complicated analysis to evaluate their results. Therefore, such techniques may not be a viable alternative for widespread use in the production of viral gene transfer vector stocks.
In view of the state of the art, there remains a need for methods to efficiently evaluate the purity of stocks of viral gene transfer vector. More particularly, there is a need to evaluate the relative purity of stocks of viral gene transfer vectors, suitable for carrying large amounts of genetic information for use in practical biological applications.